Glycation of proteins forms fructosamines and advanced glycation endproducts. Glycation adducts may be risk markers and risk factors of disease development. We measured the concentrations of the early glycation adduct fructosyl-lysine and 12 advanced glycation endproducts by liquid chromatography with tandem mass spectrometric detection. Underivatized analytes were detected free in physiological fluids and in enzymic hydrolysates of cellular and extracellular proteins. Hydroimidazolones were the most important glycation biomarkers quantitatively; monolysyl adducts (N(epsilon)-carboxymethyl-lysine and N(epsilon)-1-carboxyethyl-lysine) were found in moderate amounts, and bis(lysyl)imidazolium cross-links and pentosidine in lowest amounts. Quantitative screening showed high levels of advanced glycation endproducts in cellular protein and moderate levels in protein of blood plasma. Glycation adduct accumulation in tissues depended on the particular adduct and tissue type. Low levels of free advanced glycation endproducts were found in blood plasma and levels were 10-100-fold higher in urine. Advanced glycation endproduct residues were increased in blood plasma and at sites of vascular complications development in experimental diabetes; renal glomeruli, retina and peripheral nerve. In clinical uraemia, the concentrations of plasma protein advanced glycation endproduct residues increased 1-7-fold and free adduct concentrations increased up to 50-fold. Comprehensive screening of glycation adducts revealed the relative and quantitative importance of alpha-oxoaldehyde-derived advanced glycation endproducts in physiological modification of proteins-particularly hydroimidazolones, the efficient renal clearance of free adducts, and the marked increases of glycation adducts in diabetes and uraemia-particularly free advanced glycation endproducts in uraemia. Increased levels of these advanced glycation endproducts were associated with vascular complications in diabetes and uraemia.

Accumulation of triosephosphates arising from high cytosolic glucose concentrations in hyperglycemia is the trigger for biochemical dysfunction leading to the development of diabetic nephropathy-a common complication of diabetes associated with a high risk of cardiovascular disease and mortality. Here we report that stimulation of the reductive pentosephosphate pathway by high-dose therapy with thiamine and the thiamine monophosphate derivative benfotiamine countered the accumulation of triosephosphates in experimental diabetes and inhibited the development of incipient nephropathy. High-dose thiamine and benfotiamine therapy increased transketolase expression in renal glomeruli, increased the conversion of triosephosphates to ribose-5-phosphate, and strongly inhibited the development of microalbuminuria. This was associated with decreased activation of protein kinase C and decreased protein glycation and oxidative stress-three major pathways of biochemical dysfunction in hyperglycemia. Benfotiamine also inhibited diabetes-induced hyperfiltration. This was achieved without change in elevated plasma glucose concentration and glycated hemoglobin in the diabetic state. High-dose thiamine and benfotiamine therapy is a potential novel strategy for the prevention of clinical diabetic nephropathy.

Glycation of proteins leads to the formation of early glycation adducts (fructosamine derivatives) and advanced glycation endproducts (AGEs). Formation of AGEs has been linked to the development of cataract, diabetic complications, uraemia, Alzheimer's disease and other disorders. AGEs are a group of compounds of diverse molecular structure and biological function. To characterize AGE-modified proteins used in studies of structural and functional effects of glycation, an assay was developed that surveys the content of early and advanced glycation adducts in proteins. The assay procedure involved enzymic hydrolysis of protein substrate, derivatization of the hydrolysate with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and HPLC of the resulting adducts with fluorimetric detection. Structural isomers of methylglyoxal-derived hydroimidazolone, glyoxal-derived hydroimidazolone, 3-deoxyglucosone-derived hydroimidazolone and Nδ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)-ornithine (THP) were determined for the first time. AGEs with intrinsic fluorescence (argpyrimidine, pentosidine) were assayed without derivatization. Limits of detection were 2–17pmol and levels of recovery were 50–99%, depending on the analyte. The AQC assay resolved structural and epimeric isomers of methylglyoxal-derived hydroimidazolones and THP. Hydroimidazolones, THP and argpyrimidine were AGEs of short-to-intermediate stability under physiological conditions, with half-lives of 1–2weeks. Their measurement provides further insight into the glycation process. The assay was applied to the characterization of human serum albumin minimally and highly modified by N∊-carboxymethyl-lysine and N∊-(1-carboxyethyl)-lysine.

Methylglyoxal is a potent glycating agent under physiological conditions. Human serum albumin is modified by methylglyoxal in vivo. The glycation adducts formed and structural and functional changes induced by methylglyoxal modification have not been fully disclosed. Methylglyoxal reacted with human serum albumin under physiological conditions to form mainly the hydroimidazolone N ␦ -(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (92% of total modification) with a minor formation of argpyrimidine, N ⑀ -(1-carboxyethyl)lysine, and methylglyoxal lysine dimer. When human serum albumin was modified minimally with methylglyoxal, tryptic peptide mapping indicated a hotspot of modification at Arg-410 located in drug-binding site II and the active site of albumin-associated esterase activity. Modification of Arg-410 by methylglyoxal was found in albumin glycated in vivo. Other sites of minor modification were: Arg-114, Arg-186, Arg-218, and Arg-428. Hydroimidazolone formation at Arg-410 inhibited ketoprofen binding and esterase activity; correspondingly, glycation in the presence of ketoprofen inhibited Arg-410 modification and loss of esterase activity. The pH dependence of esterase activity indicated a catalytic group with pK a ‫؍‬ 7.9 ؎ 0.1, assigned to the catalytic base Tyr-411 with the conjugate base stabilized by interaction with the guanidinium group of Arg-410. Modification by methylglyoxal destabilized Tyr-411 and increased the pK a to 8.8 ؎ 0.1. Molecular dynamics and modeling studies indicated that hydroimidazolone formation caused structural distortion leading to disruption of argininedirected hydrogen bonding and loss of electrostatic interactions. Methylglyoxal modification of critical arginine residues, therefore, whether experimental or physiological, is expected to disrupt protein-ligand interactions and inactivate enzyme activity by hydroimidazolone formation.

SummaryStudies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C . elegans : progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C . elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C . elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C . elegans lifespan.